Method and apparatus for molecular spectroscopy, particularly for the determination of products of metabolism

ABSTRACT

Method and apparatus for molecular spectroscopy, particularly for the determination of products of metabolism. 
     The absorption of infrared radiation by a specimen (2) which contains the substance to be determined is measured. The absorption is measured simultaneously at two different wavelengths (λ 1 , λ 2 ), the first wavelength (λ 1 ) being so selected that upon changes in concentration of the substance to be determined in the specimen (2) only a negligibly small change, if any, of the radiation absorption takes place, while the second wavelength (λ 2 ) lies in the region of a substance-specific absorption band of the substance to be determined. After measurement of the radiation intensities (I 1  and I 2 ) at the two wavelengths (λ 1 , λ 2 ) the signal is standardized by formation of the quotient (I 2  /I 1 ). 
     The method makes it possible to effect a quantitative reproducible measurement in the natural biological medium or in dialysates without pretreatment of the specimen and with only small amounts of substance. By suitable selection of the second wavelength (λ 2 ) a plurality of substances can be determined.

The present invention relates to a method of molecular spectroscopy,particularly for the determination of products of metabolism, in whichthe absorption of infrared radiation by a specimen containing asubstance to be determined is measured, as well as to an apparatus forthe carrying out of this method.

The method and the apparatus of the invention can be used in particularfor the determination of glucose in serum or in urine. Such adetermination is of great importance for the recognition of diabetesmellitus and as check-up on the treatment thereof. The knowndetermination by means of test strips in urine serves primarily todetect the disease. Although a well-stabilized diabetic can, in case ofregular examination of the urine, get along with a few spot checks ofblood glucose, the determination of the concentration of glucose in theblood is essentially of importance for treatment, verification oftreatment and adjustment of the daily profile.

The known methods for the determination of glucose can be divided intothree groups, namely biochemical, electrochemical and spectroscopicmethods. Of these methods, the biochemical methods are suitable only aslaboratory procedures; the electrochemical methods, to be sure, atpresent afford the most promising prospects for the development ofimplantable glucose sensors but are inferior in their precision andspecificity to the spectroscopic methods, as the biochemical methodsalso are.

The biochemical and electrochemical methods furthermore have the commondisadvantage that the specimen to be measured must be so prepared byaddition of chemicals before the actual measurement process as to formreaction products which can be detected in the measurement. Thuscontinuous measurements are not possible.

In contradistinction to the biochemical and electrochemical methods, theglucose molecule is not modified in the spectroscopic method.

In addition, there are still a few non-specific methods which arepractically no longer used. Thus, for instance, from German Pat. No. 2724 543 a method is known for determining glucose based on thelong-known polarimetry, which, however, is today rarely used because ofinsufficient specificity, it being suitable at most under favorableconditions for the determination of sugar in the urine.

A first possibility of determining products of metabolism byspectroscopic measurement is represented by laser Raman spectroscopy.The frequency of the exciter radiation is here in the visible spectralrange. For the measurement, the portion in the spectrum of the scatteredlight which is shifted towards the red is used.

In measurements in whole blood the difficulty is encountered in thismethod of measurement that, due to the hemoglobin and the otherchromophoric substances, blood exhibits strong absorption throughout theentire visible spectral range, which, to be sure, leads to areinforcement of the resonance of the Raman scattering for thesemolecules but has a high fluorescence background associated with it andthus makes the detection of non resonance-reinforced bands difficult ifnot entirely impossible. This problem, to be sure, can be solved withthe presently available possibilities of exciting the Raman scatteringwith short laser pulses in the subnanosecond range; however, thetechnical expenditure is so substantial that it will not be possible inthe foreseeable future to find any practical method with it for samplesof whole blood.

Another possibility of avoiding the disturbance by fluorescence of thechromophores consists in operating in the infrared spectral range, i.e.in recording directly the infrared spectrum of the specimen. However,since the preparation of the specimen as well as the recording andevaluation of the spectrum are time-consuming and complicated, infraredspectroscopy of the type customary up to now does not constitutecompetition for the other methods which exist.

Another spectroscopic method is the so-called attenuated totalreflectance (ATR) spectroscopy, i.e. total reflectance spectroscopy withtransversely attenuated wave.

Thus it is known, for instance, from West German Application for PatentNo. 2 606 991 to use a CO₂ laser in combination with the well-known ATRspectroscopy to determine glucose or else other products of metabolism.This can be done, however, only in pure solutions; in multi-componentsystems or even in whole blood this method necessarily fails, due tobasic difficulties.

Thus, for instance, the Lambert-Beer law for ATR spectroscopy appliesonly for an absorption coefficient within the range of 0.1 and less andfor an angle of incidence of approximately 60° or more, referred to thematerials customary in IR, and furthermore only in case of sufficientlylarge wave lengths, and even then only approximately for isotropicspecimens. Even upon in-vitro measurements in whole blood thereforedifficulties would already arise caused, for instance, also by theprotein adsorption on the reflection surfaces, which convert the systeminto anisotropy.

In spectroscopic measurements in the infrared spectral region, theabsorption contribution of the solvent or embedment material for thesubstance to be determined is customarily compensated for by the use ofa reference ray in addition to the actual measurement ray, infraredradiation of the same wavelength being used in both rays. The requiredreproducibility of the measurement in the reference ray, however,requires pretreatment of the specimen and is not directly applicable tosubstances to be examined which are present in the natural biologicalmilieu.

The object of the present invention is now to provide a method fordetermining products of metabolism which makes possible quantitativereproducible measurement in the natural biological milieu withoutpretreatment of the specimen and therefore also without consumption ofchemicals, and in addition requires only small amounts of substance.

Proceeding from the basis of the method described in the preamble toclaim 1, this object is achieved in the manner that measurement iseffected simultaneously at two different wavelengths of the infraredradiation, the first wavelength being selected in such a manner thatupon changes in concentration of the substance to be determined in thespecimen no change in the infrared absorption or only a negligiblechange occurs, while the second wavelength is so selected that it liesin the region of a substance-specific absorption band of the substanceto be determined and that the quotient of the absorption values measuredat these two wavelengths is formed.

The method of the invention can therefore be characterized as atwo-wavelength method in which the first wavelength lies in a"quasi-isosbestic" range while the other wavelength lies in the range ofa substance-specific absorption band, in such a manner that in the caseof this latter wavelength a change in absorption is caused only by achange in concentration of the substance examined. In general,wavelengths of these properties can be readily selected on basis ofexisting infrared absorption spectra of the specimens in question sincein addition to the substance-specific absorption bands there arequasi-isosbestic ranges also in multi-component mixtures.

By means of the method of the invention it is possible, for instance, todetermine glucose in human whole blood rapidly and dependably. It isalso possible to measure other products of metabolism such as, forinstance, ethyl alcohol, urea, uric acid, creatinine, peptidedecomposition products, polystyrol and lipids in the blood or in otherbody liquids. The measurement can also be effected in dialysates, i.e.in liquids which are not body fluids but contain metabolism products.One such dialysate is, for instance, the liquid used for dialysis inkidney patients.

The method of the invention can be carried out by determining theabsorptions at the two different wavelengths by means of transmissionspectroscopy or by means of reflectance spectroscopy, and particularlyATR spectroscopy. Transmission measurements have the advantage overmeasurements by means of ATR spectroscopy, for instance in the case ofthe determination of glucose in whole blood, that the Lambert-Beer lawapplies here and that they can be calibrated by biochemicalabsolute-determination for the obtaining of quantitative measurementvalues.

The sample to be measured is advisedly present in the form of a smear orfilm on a disposable or throw-away specimen support of plastic. Thesupport material is so selected that it has only a slight absorption ofits own in the region of the first and second wavelengths. The specimensupport can be developed as a flat microscope slide, but also possiblyas a flow-through or trough cell.

As already mentioned, there are also quasi-isosbestic regions inmulti-component mixtures so that a wavelength can be found for whicheven upon changes in concentration of several substances in the specimenno change in the infrared radiation absorption takes place or only anegligibly small change. In this way, several substances in the specimencan be determined one after the other in one and the same apparatus bychanging the first wavelength in such a manner that it lies in each casewithin the region of a substance-specific absorption band. In each casethe measurement signal is standardized, the specimen itself serving asreference.

An apparatus for the carrying out of the method of the invention ischaracterized by a source of infrared radiation for the production of aninfrared radiation beam which consists of infrared radiation of at leasta first and a second wavelength, the first wavelength being so selectedthat upon changes in concentration of the substance to be determined inthe specimen no change in the infrared radiation absorption takes placeor else only a negligibly small change while the other wavelength is soselected that it lies in the range of a substance-specific absorptionband of the substance to be determined, by a detector device for theseparate measurement of the absorption values of the infrared radiationof the different wavelengths, and by a division circuit arranged behindthe detector device for forming the quotient of the absorption valuesascertained.

The source of infrared radiation can contain a strong continuum radiatoror one or more lasers, in which connection gas or solid lasers can beused. For the separating out of the two wavelengths used for themeasurement interference filters can be provided or suitably developedbeam splitters.

It is advantageous to use only one detector on the reception end and tomake the radiation beams of different wavelength distinguishable bydifferent modulation.

By the formation of the quotient of the absorption values which aremeasured for the two wavelengths one obtains a standardized measurementsignal, the specimen itself serving as reference.

Further features of the invention are set forth in the subordinateclaims.

The invention will be described in further detail below with referenceto FIGS. 1 to 12 of the accompanying drawings, in which:

FIG. 1 is an infrared absorption spectrum of water;

FIG. 2 is an infrared absorption spectrum of heparin;

FIG. 3 is an infrared absorption spectrum of D-glucose;

FIG. 4 is an infrared absorption spectrum of dried human whole blood theglucose content of which is within the normal physiological range;

FIG. 5 is an infrared absorption spectrum of dried human whole bloodwhich has been enriched with D-glucose to such an extent that theglucose content lies within the pathological range;

FIG. 6 shows a first embodiment of an apparatus in accordance with theinvention;

FIG. 7 shows a second embodiment of an apparatus;

FIG. 8 shows a third embodiment of the invention with a modified sourceof infrared radiation;

FIG. 9 shows a fourth embodiment of the invention;

FIG. 10 shows a fifth embodiment of the invention;

FIG. 11 shows transmission curves of two single-band interferencefilters such as used in the embodiments of FIGS. 6, 7 and 9; and atransmission curve of a double-band interference filter such as used inFIG. 8; and

FIG. 12 shows the transmission curves of a wavelength-selective beamsplitter such as provided in the embodiments of FIGS. 7 and 9.

The selection of the two wavelengths used for the measurement will bedescribed in connection with FIGS. 1 to 5, using the determination ofglucose as example. One essential difficulty in this determination inwhole blood or urine is that body fluids consist of water to a highpercentage. Water, however, has an absorption coefficient of 700 cm⁻¹ inthe region of the glucose absorption in the infrared. Glucose, on theother hand, has an absorption coefficient of merely about 0.1 cm⁻¹ inthis range. This difficulty, which is present in general in IRmeasurements in aqueous solutions, is customarily avoided by a so-calleddouble-beam method. In it the absorption of the solvent or the embedmentmeans is compensated for by a reference ray path in which a specimenwhich consists only of water is present. By the use of lasers instead ofthe conventional sources of light this problem can be reduced further.

The double-beam method, however, has the fundamental disadvantage that acomplicated preparation of the specimen is necessary in order to makemeaningful use of the reference ray path possible.

In the method of the invention, a double-wavelength method is usedinstead of the double-beam method. In it, the absorption of the specimenis determined simultaneously at two different wavelengths. The twowavelengths are so close to each other that dispersive effects can bekept small. This permits of measurement in the biological medium withoutcomplicated and tedious prior treatment of the specimen.

The first wavelength λ₁ is selected in such a manner that upon changesin concentration of the substance to be examined in the specimen only anegligibly small change of the absorption, if any, takes place, i.e. λ₁should lie at the "isosbestic point" or in a "quasi-isosbestic" region.

Such a quasi-isosbestic region is indicated in FIGS. 4 and 5 by thelines 41 and 42. It can be seen that in this region upon a change of theglucose concentration there is only a negligibly small change in theabsorption. This absorption, therefore, corresponds to the fundamentalabsorption of the specimen and is suitable for the standardization ofthe measurement signal.

The line 41 corresponds to the value 940 cm⁻¹ and the line 42 to thevalue 950 cm⁻¹.

The second wavelength λ₂ is selected in such a manner that it lies on asubstance-specific absorption band. This condition is satisfied bywavelengths in the region between the two wavelengths designated 51 and52 in FIGS. 1 to 5. Line 51 corresponds to a value λ₂ of 1090 cm⁻¹ andline 52 to 1095 cm⁻¹. From FIGS. 3 and 2 it can be seen that the regionindicated lies on an absorption band of glucose and at the same time,however, in the region of minimal absorption for heparin. Themucopolysaccharide heparin is present in whole blood in a relativelyhigh percentage in the basophilic leucocytes. In the normal case, theinfluence is not too high, about 0.5%, but in borderline pathologicalsituations with increased number of leucocytes or else afteradministration of heparin as an anticoagulant a substantial disturbanceof the glucose measurement can take place. By the selection of thewavelength λ₂ shown, disturbance of the measurement by heparin in thespecimen is avoided.

If a CO₂ laser is used as source of radiation the λ₂ lines correspond tothe laser lines R (40) to R(52) and the λ₁ wavelength range lies betweenthe CO₂ laser lines P(14) to P(26). The wavelength selection isadvisedly effected by suitable interference filters which can beproduced in these regions in accordance with the prior art with a halfwidth of about 5 cm⁻¹. Instead of a CO₂ laser there may also be used asemiconductor laser, for instance a Pb_(1-x) --Sn_(x) --Te-- or a Ramanlaser, or else a continuum irradiator with frequency selection. Thedetection of the measurement signal is effected with the methods andapparatus customary in the prior art.

The embodiments of devices for the determination of products ofmetabolism shown in FIGS. 6 to 10 can be used both for transmissionspectroscopy and for ATR total reflect and spectroscopy withtransversely attenuated infrared light waves. In this connection, thespecimen carrier 1, which has been only schematically indicated, in oron which the specimen 2 is provided is made preferably either asmicroscope slide or as cell from, for instance, a copolymer ofpolyethylene and polypropylene, depending on whether the specimen 1 isirradiated vertically or horizontally.

Each of the embodiments shown has a source of infrared radiation 3 and adetector device 4. Within the infrared radiation beam 5 which forms theray path between the source 3 and the detector 4 there is located thespecimen carrier 1 bearing the specimen 2 to be measured.

The source of infrared radiation 3 is developed in such a manner that itproduces the infrared radiation beam 5 which consists of infraredradiation of a first and a second wavelength λ₁ and λ₂ respectively. Thedetector device 4 is developed in such a manner that it measuresseparately the absorption or intensity values of the infrared radiationof the different wavelengths λ₁ and λ₂. Behind the detector device 4there is provided a divider circuit 6 to which the two signals I₁ and I₂determined by the detector device 4 are fed and which forms the quotientQ=I₂ /I₁, in which I₂ is the absorption or intensity value correspondingto the wavelength λ₂ while I₁ is the absorption or intensity valuecorresponding to the wavelength λ₁. Behind this divider circuit 6 thereis an indicating and/or recording device 7 which can be developed, forinstance, as recorder, printer or digital or analog display instrumentor the like.

In the embodiment shown in FIG. 6, the infrared radiation source 3 has astrong continuum radiator 8 such as, for instance, a Globor or Nernstrod. By means of a diaphragm 9 a first and a second infrared radiationbeam 10 and 11 respectively are stopped out. For the selecting of thetwo wavelengths λ₁ and λ₂ from the continuum spectrum of the source ofradiation 8 a first interference filter 12 is arranged in the ray pathof the individual ray 10, it passing essentially only radiation of thewavelength λ₁, while in the ray path of the second individual ray 11there is arranged a second interference filter 13 which passessubstantially only radiation of the wavelength λ₂.

The basic course of the transmission function of the interferencefilters 12, 13 is shown in the upper and middle parts of FIG. 11 inwhich the transparency T in percent is plotted over the wavelength λ inμm. The half-width value Δλ should be smaller than or equal to 1% of thecorresponding wavelength λ₁ or λ₂ which is to be transmitted.

The two individual rays 10, 11 are combined by mirrors 14 and awave-length selective beam divider 15 to form a single infraredradiation beam 5 after they have previously been modulated by a chopper16 with different frequencies f₁ (modulation frequency of the infraredradiation beam 10) and f₂ (modulation frequency of the infraredradiation beam 11) respectively. The fact that different frequenciesresult is indicated in the manner that the chopper disk 17 of thechopper 16 cuts the infrared radiation beam 11 at a place located closerto the axis of rotation 18 of the chopper 16 than the infrared radiationbeam 10, and the chopper blade for the two beams (10, 11) has adifferent number of segments.

The transmission function of the wavelength-selective beam splitter 15is shown in FIG. 12 in which the transparency T and the reflectivity Rare plotted, in each case in percent, over the wavelength in μm, and thewavelengths λ₁ and λ₂ entered.

The radiation beam 5 passes through the specimen 2, which may forinstance be a smear of a drop of blood, on a specimen holder 1 developedas a microscope slide. This slide may consist, for instance, of acopolymer of polyethylene and polypropylene. After passage through thespecimen 2, the infrared radiation beam 5 passes into the detectordevice 4 and comes here against a single detector 19 which haspractically the same sensitivity in the range in which the wavelengthsλ₁ and λ₂ are. For example, the detector 19 can be a pyroelectricreceiver, such as for instance a triglycin-sulfate crystal, referred toin abbreviated fashion also as TGS crystal. This practically identicalsensitivity is necessary in order that the following electronic systemcan be operated with the same time constants. The output signal of thedetector 19 is fed to two parallel lock-in amplifiers 20, 21(phase-sensitive rectifiers with possibly amplifier behind same), one ofwhich is tuned to the modulation frequency f₁ of the infrared radiationportion of the wavelength λ₁ and the other to the modulation frequencyf₂ of the infrared radiation portion of the wavelength λ₂. At the outputof the lock-in amplifier 20 the abovementioned intensity value I₁ or asignal proportional to it is obtained, while at the output of thelock-in amplifier 21 there is available the above-indicated intensityvalue I₂ or a signal proportional to it. These two signals are fed intothe following division circuit 6 which produces therefrom thestandardized concentration-proportional signal Q=I₂ /I₁.

In the embodiment shown in FIG. 7, the chopper 16 is so arranged anddeveloped that it modulates two individual rays 10 and 11 with the samechopping frequency f. Accordingly a second wavelength-selective beamsplitter 22 is provided in the corresponding detector device 4 and itdivides the sole infrared beam 5 impinging upon it into a firstindividual beam 23 of the wavelength λ₁ and a second individual beam 24of the wavelength λ₂. The first individual beam 23 comes onto a detector25 and the second individual beam 24 strikes a second detector 26. Thesedetectors 25, 26 may be of the same type as the detector 19 of FIG. 6.

The lock-in amplifiers 27 and 28 arranged behind the two detectors 25,26 respectively, both of which amplifiers are tuned to the modulationfrequency f, in their turn produce at their outputs intensity values I₁and I₂ respectively from which the above-indicated quotient Q is formedin the division circuit 6.

FIG. 8 shows an embodiment in which only a single radiation beam, namelythe infrared radiation beam 5, is stopped-out by the diaphragm 9 fromthe radiation of the source of radiation 8, which is also developed ascontinuum radiator. This beam passes through a double-band interferencefilter 29 and the chopper disk 17 of a chopper 16 and then passesthrough the specimen 2. The transmission curve of this double-bandinterference filter 29 is shown in the lower part of FIG. 11 and, as canbe noted from a comparison with the middle and upper parts of FIG. 11,represents a combination of the transmission curves of the twointerference filters 12 and 13 which are used in the embodiments ofFIGS. 6 and 7.

The detector device 4 in FIG. 8 can be developed in the manner shown inFIG. 7.

FIG. 9 shows an embodiment in which the infrared radiation source 3 hasas source of light a laser 8, for instance a CO₂ laser or a Raman laserwith multi-line emission. This laser is provided with a beam-wideningdevice 30. By means of the diaphragm 9 a single radiation beam, namelythe infrared radiation beam 5 is stopped-out and is modulated before itpasses through the specimen 2, by means of a chopper 16 with a choppingfrequency f.

The detector device 4 is essentially of the same construction as shownin FIG. 7; however, for purposes of wavelength selection a firstinterference filter 12 is arranged in the ray path of the firstindividual beam 23 between the wavelength-selective beam splitter 22 andthe first detector 25 so that the latter receives only infraredradiation of the wavelength λ₁ while a second interference filter 13which passes only radiation of the wavelength λ₂ is provided in the raypath of the second individual beam 24 between the wavelength selectivebeam splitter 23 and the second infrared radiation detector 26.

The embodiment of FIG. 9 can also be modified in such a manner that bythe splitting up and recombination of the radiation beam emerging fromthe beam widening device 30, with selection of the wavelengths λ₁ and λ₂and of a double-frequency modulation by means of a chopper 16 inaccordance with the embodiment of FIG. 6, it is possible to use adetector device 4 of the type shown in FIG. 6 which requires only asingle detector 19.

In FIG. 10 there is shown an embodiment in which two monochromaticlasers 8 are provided, one of which has the emission wavelength λ₁ andthe other the emission wavelength λ₂. These lasers 8 can, for instance,be Pb_(1-x) Sn_(x) Te lasers so that, after passage of the laserradiations through the beam-widening device 30, a first individual beam10 is produced by the diaphragm 9, which beam contains only infraredradiation of the wavelength λ₁, as well as a second individual beam 11which contains only infrared radiation of the wavelength λ₂. These twoindividual beams are modulated in a manner similar to that described inFIG. 6 with two different chopping frequencies f₁ and f₂ respectivelyand are combined to form a common infrared radiation beam 5 by a mirror14 and a wavelength-selective beam splitter 15. The detector device 4can in this embodiment be of the type shown in FIG. 6 but it can also bedeveloped in the manner shown in FIG. 7 provided that the two individualbeams 10, 11 in FIG. 10 are modulated with the same chopping frequencyf.

The specimen space can also be developed as a flow-through cell of smalllayer thickness. This is particularly of interest when the method is tobe used for continuous measurement, for instance upon dialysis with anartificial kidney. In this case of use what is of interest is less theglucose determination than rather the determination of peptide fissionproducts, defect hormones, urea, uric acid and creatinine in thedialysate. The isosbestic or quasi-isosbestic region for λ₁ is in thisconnection substantially the same as in the glucose measurement but thesecond wavelength λ₂ must be selected in each case in accordance withthe substances. The optical and electrical construction of theevaluation apparatus itself is selected in accordance with one of theembodiments shown.

It is also possible to determine several substances simultaneously withthe method of the present invention. In that case more than twowavelengths must then be radiated simultaneously through the specimen,one of these wavelengths lying in an at least quasi-isosbestic regionand the others being selected in each case adapted to the substances tobe investigated. Thus it is readily possible, for instance, to determineglucose, ethyl alcohol, uric acid and creatinine simultaneously with theuse of five different wavelengths. The standardization is effected ineach case by the fifth wavelength, which lies in a quasi-isosbesticrange, for instance in the range shown in FIGS. 1 to 5.

I claim:
 1. A method for the spectroscopic determination of glucoseconcentration in an unpretreated multi-component specimen selected fromthe group of whole blood and urine which comprises the steps of:(a)simultaneously measuring the absorption values of infrared radiation bysaid specimen at a first wavelength lying within the infrared spectralrange of 940 to 950 cm⁻¹ and a second wavelength lying within theinfrared spectral range of 1090 to 1095 cm⁻¹ ; and (b) standardizing themeasurement by forming the quotient of the absorption values of thefirst and second wavelengths:whereby the glucose concentration isproportional to the absorption value measured at second wavelength, andthe absorption value measured at said first wavelength is essentiallyindependent of said concentration.
 2. A method according to claim 1,wherein the absorption value of said first and second wavelengths isdetermined by means of transmission spectroscopy.
 3. A method accordingto claim 1, wherein the absorption value of said first and secondwavelengths is determined by means of reflection spectroscopy.
 4. Amethod according to claims 2 or 3, wherein the specimen is provided as asmear or film.
 5. A method according to claims 2 or 3, wherein theinfrared radiation of the first and second wavelengths are modulated atdifferent frequencies.
 6. An apparatus for determining the concentrationof glucose in an unpretreated multi-component specimen selected from thegroup of whole blood and urine, comprising means for generating aradiation beam, said beam comprising a first wavelength lying within theinfrared spectral range of 940 to 950 cm⁻¹ and a second wavelength lyingwithin the infrared spectral range of 1090 to 1095 cm³¹ 1 ; means forpositioning the specimen in said radiation beam; a detector means forthe separate but simultaneous measurement of the absorption values ofthe infrared radiation at said first and second wavelengths: and adivision circuit adapted to form the quotient of the absorption valuesof said first and second wavelength.
 7. An apparatus according to claim6, wherein the means for generating said radiation beam comprising saidfirst and second wavelengths includes a source of infrared radiation; adiaphragm for stopping-out first and second individual radiation beams;a first interference filter positioned within the path of the firstindividual radiation beam, said first interference filter being adaptedto pass only infrared radiation of said first wavelength; a secondinterference filter positioned within the path of the second individualradiation beam, said second interference filter being adapted to passonly infrared radiation of said second wavelength; and means fordeflecting and combining said first and second individual beams intosaid single beam comprising said first and second wavelength prior topassing through said specimen.
 8. An apparatus according to claim 7,which further includes a chopper positioned within the ray path of thefirst and second individual radiation beams adapted to modulate thefirst individual ray beam with a first frequency and to modulate thesecond individual radiation beam with a second frequency.
 9. Anapparatus according to claim 8, wherein the detector means includes asingle infrared radiation detector the output of which is fed into firstand second parallel lock-in amplifiers said first amplifier being tunedto the first frequency and said second amplifier being tuned to thesecond frequency, and the outputs of the lock-in amplifiers beingconnected to the division circuit.
 10. An apparatus according to claim6, wherein said means for positioning said specimen is a disposablespecimen support constructed of a plastic which has only a slightabsorption of its own within the range of the first and secondwavelengths.
 11. An apparatus according to claim 6, wherein a strongcontinuum radiator is provided to generate said infrared radiation. 12.An apparatus according to claim 6, wherein one or more lasers areprovided to generate said infrared radiation.
 13. An apparatus accordingto claim 6, wherein the means for generating said radiation beamcomprising said first and second wavelengths includes a source ofinfrared radiation; a diaphragm for stopping-out a single infraredradiation beam; and a double-band interference filter which passes onlyinfrared radiation of said first and second wavelengths.